A naturally-occurring mutation in Cacna1f in a rat model of congenital stationary night blindness

Purpose To identify the gene mutation responsible for a previously described rat model of X-linked congenital stationary night blindness (CSNB). Methods Rat orthologous genes for Nyx and Cacna1f were isolated from retina through rapid amplification the cDNA ends (RACE) and examined for mutations. Electroretinograms were used to identify affected animals. Results The rat Nyx cDNA spans 1,971 nucleotides and encodes a protein of 476 amino acids (GenBank: DQ393414). The rat Cacna1f cDNA spans 6,076 nucleotides and encodes a protein of 1,980 amino acids (GenBank: DQ393415). A c.2941C>T (p.R981Stop) mutation in Cacna1f was found in affected rats. Immunochemistry study showed labeling for rod bipolar and horizontal cells were reduced in affect retinas. For affected rats, b-wave and oscillatory potentials of scotopic ERG were absent, and b-wave of photopic ERG was clear but obviously reduced. Conclusions The Cacna1f mutation identified in the rat model of CSNB was predicted to lead to a protein product that is shortened by 999 amino acids, indicating that this is a model for the incomplete subtype of human X-linked CSNB (CSNB2). This rat model will be useful for defining the pathophysiological properties of this human disorder.

described previously [20]. After 10 h dark adaptation, rats were anesthetized intraperitoneally with ketamine (70 mg/kg, Sigma, Saint Louis, MO) and xylazine (10 mg/kg, Sigma). The pupils were dilated with 0.5% tropicamide, and animals were secured to a platform with a heating pad to maintain the body temperature. ERGs were recorded from the corneal surface using a silver-chloride electrode loop that made contact through a layer of 1% methylcellulose. Stainless steel needle electrodes placed in the cheek served as reference leads while those placed in the tail acted as ground leads. ERGs were recorded by a commercial system (RETIport; Roland Consult GmbH, Brandenburg, Germany) using a band pass of 0.5 to 1000 Hz. Strobe stimulus flashes were delivered in a Ganzfeld, and neutral density filters were used to control stimulus intensity.
A dark-adapted intensity series was recorded first, using a stimulus range of -2.5 to 0.5 log cd s mm -2 . Interstimulus intervals increased from 15 s at the lower flash intensities, to 2 min at the highest flash levels. A steady adapting field (1.3 log cd mm -2 ) was then presented within the Ganzfeld. After a 10-minute period of light adaptation, cone ERGs were elicited by flash stimuli superimposed against the adapting field. Cone ERGs were recorded in response to stimuli ranging from -2.5 to 0.5 log cd s mm -2 . In each case, the responses to 25 consecutive flashes presented at 2.1 Hz were averaged.
Isolation of the rattus norvegicus Nyx and Cacna1f fulllength cDNA: Smart Race technology (Clontech, Mountain View, CA) was used to amplify the full-length cDNA of Nyx and Cacna1f from the rat. Total RNAs were isolated from retinas of affected and control rats with Trizol reagent (Invitrogen, Frederick, MD) according to manufacturer's protocol. Primers were designed from predicted rat Nyx sequence (GenBank: NM_001100967) and available Cacna1f sequence (GenBank: NM_053701) in Table 1. The PCR products were subcloned into the pMD-18T vector (Takara, Dalian, China) and transformed into E.coli, and sequenced.
Bioinformatics: The open reading frame (ORF) and translated amino acid sequences were predicted by the National Center for Biotechnology Information's (NCBI) ORF Finder and DNAStar 2.0 program. The nucleotide and amino acid sequences were aligned in NCBI's Blast program to search for sequence matches. The chromosomal location and extronintron structure were analyzed in the NCBI's Genomic Biology. The sequences to be analyzed were retrieved from GenBank by NCBI's Entrez system. Multiple alignments were performed with Clustal X (1.8) program. Motif searches were carried out with ExPASy and Smart program.
Mutation screening: To identify whether a mutation in Nyx or Cacna1f gene was present in the CSNB-like rat, we

Gene
Purpose Full length cDNA of rat Nyx and Cacna1f were isolated by 5' and 3' rapid amplification the cDNA ends. Primers were designed according to available sequences and manufacturer's protocol.
designed primers to amplify fragments encompassing the fulllength cDNA from both control and affected rat ( Table 2). The PCR products were subcloned into TA vectors (pMD-18T, Takara) and sequenced as described in the previous section. Histology: After 10-week old control and affected rats were killed by CO 2 (5-10 min, in a sealed chamber), the eyes were enucleated, dissected along the ora serrata, and posterior eyecups were fixed in 4% paraformaldehyde at 4 °C for overnight. The eyecups were then rinsed in phosphate buffer (PB), dehydrated through a graded series of ethanol washes, and embedded in paraffin. A microtome was used to cut 5 µm thick sections, which were mounted onto slides and stained with hematoxylin and eosin for anatomy analysis.
Immunochemistry: Sections were rehydrated, and 3% H 2 O 2 in 40% methanol was used to block the endogenous peroxidase. After sections were incubated in blocking solution (10% goat serum, Boster) for 30 min, solutions were replaced and sections were incubated with primary antibody diluted in blocking solution at 4 °C for overnight. Primary antibodies used were a 1:5000 of anti-protein kinase Cα (PKC, Sigma) and a 1:3000 dilution of anti-calbindin D-28K (Chemicon, Southampton, UK). After washing with PB, sections were incubated in a 1:1000 dilution of biotin-conjugated secondary antibody (Boster, Wuhan, China) for 1 h. After a wash in PB, sections were incubated in an avidin-biotin peroxidase com-plex (Boster) for 30 min. Sections were rinsed in PB and immunoreaction was visualized with a diaminobenzidinenickel solution (Boster) as the chromogen. The reaction in PB, before slides were coverslipped and images were captured digitally on light microscopy. Figure 1A presents ERGs recorded from representative control (left) and mutant (right) animals under dark-adapted conditions. Under dark-adapted conditions, ERGs of mutant rats lacked distinct b-waves or oscillatory potentials throughout the range of stimulus intensities. In response to high stimulus intensities, mutant rats generated a clear a-wave. In comparison to control responses, a-waves of mutant rats were significantly reduced in amplitude. Figure  1B,C show intensity-response functions for dark-adapted aand b-waves of ten control and ten affected rats.   Figure 2A presents representative ERGs recorded under light-adapted conditions. In comparison to control responses, light-adapted ERGs of mutant rats were significantly smaller in amplitude and delayed in peak time. Figure 2B,C show intensity-response functions for the cone ERGs.

Electrophysiology:
Isolation of rat Nyx and Cacna1f cDNA: Full-length rat Nyx cDNA spans 1,971 nucleotides (GenBank: DQ393414). Compared with the computational predicted one (GenBank: NM_001100967), this sequence has complete 5' and 3' UTR. When aligned with the rat genome, it was predicted to locate in Xq12 and contain 3 exons. The ORF was confined to exon 2 and 3 as in human [12,13], mouse [21,22], chic [23], and zebrafish [24] genes, and encodes a protein of 476 amino acids with a predicted molecular weight of 52.5 kDa. The nucleotide sequence is 86% and 93% identical to human (GenBank: NM_022567) and mouse (GenBank: NM_173415) sequences, respectively. The translated amino acid sequence is 84% and 95% identical to human (GenBank: NP_072089) and mouse (GenBank: NP_775591) sequences, respectively ( Figure 3). Computational protein motif analysis of rat Nyx predicted a characteristic domain structure: the N-terminal putative sig-nal sequence, the core segment consists of 11 leucine-rich regions (LRRs) flanked by two cysteine-rich LRRs (LRRNT and LRRCT). Unlike human, the C-terminal GPI membrane anchor was not predicted in rat sequence. Only a potential cleavage site was found at C-terminal. Identity was much higher in the LRR core segment than in the signal sequence and C terminal among the species. Recent work has suggested both human and mouse nyctalopin are membrane-bound extracellular proteins with function conserved [25], and orthologous nyctalopin proteins may have different mechanisms of cell membrane attachment [26].
Full-length of rat Cacna1f cDNA spans 6,076 nucleotides (GenBank: DQ393415) and has complete 3'-UTR compared with available one (GenBank: NM_053701). When aligned with the rat genome, it was predicted to locate in Xq13 and contain 49 exons. The ORF was confined to all of the exons and encodes a protein of 1980 amino acids with a predicted molecular weight of 220.0 kDa. The rat Cacna1f shares 88% and 95% identity with human (Genbank: NM_053701) and mouse (GenBank: NM_019582) sequences, respectively. The translated amino acids shares 91% and 97% with human (GenBank: NP_005174) and mouse (Genbank: NP_062528) sequences, respectively ( Figure 4). Computational protein motif analysis showed four homologous domains of ion transport protein (Ion_trans), each containing six transmembrane alpha helices. The transmembrane segments were best conserved among species, and the most disparate regions were C terminal and cytoplasmic loop between domains 2 and 3 [27].
Mutation analysis: To identify the mutation responsible for CSNB rats, we amplified fragments encompassing Nyx or Cacna1f from cDNAs isolated from the retinas of control and mutant rats, which were identified by ERG analysis. Sequence analysis revealed a point mutation of C to T at position 2941, which changes codon 981 from arginine (CGA) to a stop codon (TGA). This R981Stop point mutation was predicted to lead to a version of protein shortened by a total of 999 amino acids, and missing the C-terminal and, in particular, part of the third and all of the fourth ion transport domains.
To confirm that R981Stop was casually associated with the phenotype, we analyzed 24 rats obtained in our breeding pedigree ( Figure 5). After ERGs were used to determine the phenotype, the mutant position was amplified from each animal's retinal cDNA. All seven affected rats were found to carry the c.2941C>T mutation in the retinal cDNAs, while no rat with a normal ERG carried this mutation.
Histology and immunochemistry: Histology showed that retinal structures were similar in both control and affected rats ( Figure 6A,B). Labeling of control retinas with PKCα showed dendrites of bipolar cells terminated in the OPL ( Figure 6C), in agreement with previous reports [28][29][30][31], whereas labeling with PKCα in affected retina was much reduced ( Figure 6D). Labeling of control rat for horizontal cells with calbindin showed staining of bodies and processes in OPL ( Figure 6E), in contrast, labeling for horizontal cells in affected retinas were rare ( Figure 6F). In affected retinas, no extension to outer nuclear layer for dendrites of rod bipolar cells and horizontal cells were observed.

DISCUSSION
This study found that the rat mutant identified by ERG recordings [19] carried a mutation in the Cacna1f gene, while Nyx is normal. As a consequence, the rat model provides a new model for CSNB2, which also involves Cacna1f mutations. The protein encoded by Cacna1f is the α 1F subunit of voltage-gated L-type calcium channels, which appear to be expressed only in the retina [12]. Immunohistochemical analysis has localized this subunit to the ribbon active zones in rod photoreceptor terminals [32]. The visual signal generated by rod and cone photoreceptors in response to light is transmitted to second order neurons through glutamate released at ribbon synapses located in the rod and cone terminals [33]. Thus, a defect in α 1F would be expected to severely diminish postreceptoral transmission of the visual signal, which is clearly seen in all patients with CSNB2 and in the available animal models.
There are two mouse models for CSNB2. Mansergh et al [30] described the phenotype of a knock-out model (Cacna1f -/-), while Chang et al [31] detailed a naturally-occurring mutant (nob2) identified, like the rat model under consideration, through fortuitous ERG studies. The rat phenotype appears to be an intermediate between these two mouse models. Under dark-adapted conditions, ERG b-waves and oscillatory potentials are essentially absent (Figure 1). This resembles the phenotype of Cacna1f -/mice [30] but not of the nob2 mouse [31]. Under light-adapted conditions, affected rats generated clear cone ERGs of reduced amplitude (Figure 2). This phenotypic feature resembles more closely the results obtained in nob2 mice [31] than of Cacna1f -/mice [30]. Immunochemistry showed that labeling for both rod bipolar cells and horizontal cells in affected retinas were reduced, especially for horizontal cells. This indicates that presence and activity of voltagegated L-type calcium channels are essential for development of second-order neurons, such as bipolar and horizontal cells. Contrary to observations made in Cacna1f mutant mouse models, neither rod bipolar nor horizontal cells dendrites were observed to extend beyond the OPL in the rat. Given the range of phenotype seen in these mouse models and in human patients with Cacna1f mutations [14,15], the Cacna1f rat model will provide an additional animal model with which to understand the relationship between Cacna1f mutations and retinal phenotypes.